The present invention relates to fabricating protein microarrays in general and in particular to a method that utilizes a gelatin-based substrate wherein the gelatin surface is modified to improve specific attachment of biological molecules.
The completion of Human Genome project spurred the rapid growth of a new interdisciplinary field of proteomics which includes: identification and characterization of complete sets of proteins encoded by the genome, the synthesis of proteins, post-translational modifications, as well as detailed mapping of protein interaction at the cellular regulation level.
While 2-dimensional gel electrophoresis in combination with mass spectrometry still remains the dominant technology in proteomics study, the successful implantation and application of DNA microarray technology to gene profiling and gene discovery have prompted scientists to develop protein microarray technology and apply microchip based protein assays to the field of proteomics. For example, in WO 00/04382 and WO 00/04389, a method of fabricating protein microarrays is disclosed. A key element in the disclosure is a substrate consisting of a solid support coated with a monolayer of thin organic film on which protein or a protein capture agent can be immobilized.
Nitrocellulose membrane was widely used as a protein blotting substrate in Western blotting and enzyme linked immunosorbent assay (ELISA). In WO 01/40312 and WO 01/40803, antibodies are spotted onto a nitrocellulose membrane using a gridding robot device. Such spotted antibody microarrays on a nitrocellulose membrane substrate have been shown to be useful in analyzing protein mixture in a large parallel manner.
In WO 98/29736, L. G. Mendoza et al. describe an antibody microarray with antibody immobilized onto a N-hydroxysuccinimidyl ester modified glass substrate. In U.S. Pat. No. 5,981,734 and WO 95/04594, a polyacrylamide based hydrogel substrate technology is described for the fabrication of DNA microarrays. More recently, in Anal. Biochem. (2000) 278, 123-131, the same hydrogel technology was further demonstrated as useful as a substrate for the immobilization of proteins in making protein microarrays.
In the above cited examples, the common feature among these different approaches is the requirement of a solid support that allows covalent or non-covalent attachment of a protein or a protein capture agent on the surface of said support. In DNA microarray technology, a variety of surfaces have been prepared for the deposition of pre-synthesized oligos and PCR prepared cDNA probes. However, unlike DNA, proteins tend to bind to surfaces in a non-specific manner and, in doing so, lose their biological activity. Thus, the attributes for a protein microarray substrate are different from those for a DNA microarray substrate in that the protein microarray substrate must not only provide surface functionality that are capable of interacting with protein capture agents, but must also resist non-specific protein binding to areas where no protein capture agents have been deposited.
Bovine serum albumin (BSA) has been demonstrated to be a useful reagent in blocking proteins from non-specific surface binding. Polyethylene glycol and phospholipids have also been used to passivate surfaces and provide a surface resistant to non-specific binding. However, all of these methods suffer disadvantages either because surface preparation takes a long time or because the method of surface modification is complex and difficult, making the method less than an ideal choice for large scale industrial manufacture.
Hence, there is still need to discover other low cost and readily manufacturable materials that serve as a matrix on a solid support for the attachment of protein capture agents. The art needs a substrate with chemical functionality for the immobilization of protein capture agents, but such substrate must not bind proteins to areas on the gelatin surface that are without immobilized protein capture agents.
The present invention seeks to solve some of the problems discussed above by providing:
A gelatin-based substrate for fabricating protein arrays, the substrate comprising: gelatin and a trifunctional compound Axe2x80x94Lxe2x80x94B; wherein A is a functional group capable of interacting with the gelatin; L is a linking group capable of interacting with A and with B; and B is a functional group capable of interacting with a protein capture agent, wherein A may be the same or different from B.
Also provided is a method of making a gelatin-based substrate for fabricating protein arrays comprising the steps of providing a support; coating on the support a composition containing gelatin; and affixing to a surface of the gelatin a trifunctional compound Axe2x80x94Lxe2x80x94B; wherein A is a functional group capable of interacting with the gelatin; L is a linking group capable if interacting with A and with B; and B is a functional group capable of interacting with a protein capture agent; wherein A may be the same or different from B.
The invention is particularly useful in fabricating protein microarrays. The invention provides a gelatin substrate with at least one surface to which certain functionalities have been affixed. Thus treated, or modified, the gelatin surface is substantially resistant to non-specific binding. Further, the functionalities are capable of interacting specifically with protein capture agents with which they come in contact. Thus, the substrate of the invention affords a high degree of specific binding between the modified gelatin surface and protein capture agents.
Gelatin substrates that have been modified according to this invention require a very low concentration of biological sample in fabricating protein microarrays when compared with unmodified gelatin substrates. Also, the gelatin substrates of the invention can be readily manufactured at low cost. The usefulness of the claimed substrate for protein attachment is demonstrated below in the examples, using several chemical modification methods and enzyme linked immunosorbent assay (ELISA).
In general, a protein microarray can be prepared by first modifying a solid support, namely the protein microarray support, followed by depositing various protein capture agents onto the modified substrate at pre-defined locations. Supports of choice for protein microarray applications can be organic, inorganic or biological. Some commonly used support materials include glass, plastics, metals, semiconductors. The support can be transparent or opaque, flexible or rigid. In some cases, the support can be a porous membrane e.g. nitrocellulose and polyvinylidene difluoride, and the protein capture agents are deposited onto the membrane by physical adsorption. However, to improve robustness and reproducibility, it is more desirable to immobilize the protein capture agents onto a substrate through chemical covalent bond.
To immobilize protein capture agents onto a solid support, the support needs to be modified by certain chemical functional agents. In general, the chemically functional agent is a bi-functional molecule which can be represented as Axe2x80x94Lxe2x80x94B, in which A and B are chemical functionalities that are capable of reacting or interacting with gelatin and protein capture agent molecules to be immobilized on the substrate and L is linkage group. Preferably, L is a diradical of such a length that the shortest throughxe2x80x94bond path between the ends that connect A to B is not greater than 10 atoms.
There are two classes of bi-functional agents: 1). homofunctional agent if A=B; and 2). heterofunctional agent if Axe2x89xa0B. Some commonly used A and B include but are not limited to, aldehyde, epoxy, hydrazide, vinyl sulfone, succinimidyl ester, carbodiimide, maleimide, dithio, iodoacetyl, isocyanate, isothiocyanate, aziridine. The linking group L comprises any reasonable combination of relatively non-labile covalently bonded chemical units sufficient to connect the two functionalities A and B. These chemical units can consists of, but are not necessarily limited to, a single bond, a carbon atom, an oxygen atom, a
sulfur atom, a carbonyl group 
a carboxylic ester group 
a carboxylic amide group 
a sulfonyl group 
a sulfonamide group 
an ethyleneoxy group, a polyethyleneoxy group, or an amino group 
where substituents X, Y, and Z are each independently a hydrogen atom, or an alkyl group of 1-10 carbon atoms; and linear or branched, saturated or unsaturated alkyl group of 1 to 10 carbon atoms (such as methyl, ethyl, n-propyl, isopropyl, t-butyl, hexyl, decyl, benzyl, methoxymethyl, hydroxyethyl, iso-butyl, and n-butyl); a substituted or unsubstituted aryl group of 6 to 14 carbon atoms (such as phenyl, naphthyl, anthryl, tolyl, xylyl, 3-methoxyphenyl, 4-chlorophenyl, 4-carbomethoxyphenyl and 4-cyanophenyl); and a substituted or unsubstituted cycloalkyl group of 5 to 14 carbon atoms such as cyclopentyl, cyclohexyl, and cyclooctyl); a substituted or unsubstituted, saturated or unsaturated heterocyclic group (such as pyridyl, primidyl, morpholino, and furanyl); a cyano group. Some solubilizing groups can also be introduced into Axe2x80x94Lxe2x80x94B and examples of these solubilizing groups include, but are not limited to, carboxylic acid, sulfonic acid, phosphonic acid, hydroxamic acid, sulfonamide, and hydroxy groups (and their corresponding salts). A and B can also be in the form of readily reactive functionalities towards crosslinkers, examples include but not limited to carboxy, amino, and chloromethyl, etc. A and B can be affinity tags that are capable of interacting non-covalently with the protein capture agents intended to be immobilized onto the substrate. For example, some commonly used tag systems include, but are not limited to, streptavidin and biotin, histidine tags and nickel metal ions, glutathione-S-transferase and glutathione. One skilled in the art should be able to create a fusion protein capture agent using recombination DNA technology and an element of tag recognition unit can be introduced into protein capture agent in this way.
Once a protein microarray substrate is modified, protein capture agents will be placed onto the substrate to generate protein microarray content. Protein capture agents are molecules which can interact with proteins in high affinity and high specificity. Typically it is desirable to have an affinity binding constant between a protein capture agent and target protein greater than 106 Mxe2x88x921. There are several classes of molecules that can be used as protein capture agents on a protein microarray. Antibodies are a class of naturally occurring protein molecules that are capable of binding targets with high affinity and specificity. The properties and protocols of using antibody can be found in xe2x80x9cUsing Antibodies; A Laboratory Manualxe2x80x9d, (Cold Spring Harbor Laboratory Press, by Ed Harlow and David Lane, Cold Spring Harbor, N.Y. 1999). Antigens can also be used as protein capture agents if antibodies are intended targets for detection. Protein scaffolds such as whole protein/enzyme or their fragments can be used as protein capture agents as well. Examples include phosphotases, kinases, proteases, oxidases, hydrolyases, cytokines, or synthetic peptides. Nucleic acid ligands can be used as protein capture agent molecules after in vitro selection and enrichment for their binding affinity and specificity to certain targets. The principle of such selection process can be found in Science, Vol. 249, 505-510, 1990 and Nature, Vol. 346, 818-822, 1990. U.S. Pat. No. 5,110,833 discloses an alternative class of synthetic molecules that can mimic antibody binding affinity and specificity and can be readily prepared by the so called Molecular Imprinting Polymer (MIP). This technology has been reviewed in Chem. Rev. Vol. 100, 2495-2504, 2000.
In practice, a protein microarray is brought into contact with a biological fluid sample, proteins in the sample will adsorb to both areas spotted with specific protein capture agents and areas without protein capture agents. Since the protein microarray is intended to be used for the measurement of specific interactions between protein capture agents on the chip with certain proteins or other molecules in the biological fluid sample, the non-specific binding of sample proteins to non-spotted area would give rise to high background noise. The term non-specific binding refers to the tendency of protein molecules to adhere to a solid surface in a non-selective manner. This high background noise resulting from the non-specific binding will interfere with reporter signals to be detected from the spotted area unless the non-specific binding is blocked in an appropriate manner. Typically, the protein microarray will be immersed in a solution containing a blocking agent to block the non-specific binding sites before its contact with the intended analyte solution. A commonly used method for blocking protein non-specific binding is to treat the surface of the substrate with a large excess of bovine serum albumin. The non-spotted surface area may also be chemically modified with polyethylene glycol (PEG), phospholipid, or poly lysine to prevent non-specific binding.
Gelatin has been used in the photographic industry as a binder for various chemical components, and the process of making high quality gelatin is well established in industry. Because gelatin is made of biological materials, it is biologically compatible with protein capture agents on the protein microarray. The gelatin coated surface provides a biologically benign surface for the immobilization of protein capture agents onto the protein microarray. As shown in this invention, gelatin also renders a surface that substantially reduce background noise that is a result of non-specific binding. Normally, gelatin is coated onto a substrate and gelation occurs through a process by which gelatin solutions or suspensions of gelatin and other materials form continuous three-dimensional networks that exhibit no steady state flow. This can occur in polymers by polymerization in the presence of polyfunctional monomers, by covalent cross-linking of a dissolved polymer that possesses reactive side chains and by secondary bonding, for example, hydrogen bonding, between polymer molecules in solution. Polymers such as gelatin exhibit thermal gelation which is of the latter type. The process of gelation or setting is characterized by a discontinuous rise in viscosity. (See, P. I. Rose, xe2x80x9cThe Theory of the Photographic Processxe2x80x9d, 4th Edition, T. H. James ed. pages 51 to 67).
The gelatin substrate described in this invention can either be coated as is on any solid support, or with one or a combination of multiple hardening agents mixed in the gel. The level of the hardening agent should be from 0 to 20 wt. %, and preferably 0.5 to 8 wt. %, of the total gelatin coated.
There are two types of gelatin: acid pretreated and alkaline pretreated. The preferred gelatin is alkaline pretreated gelatin from bovine bone marrow, but gelatin can also come from other sources. Examples include, but are not limited to, pig gelatin, fish gelatin. The bi-functional agent Axe2x80x94Lxe2x80x94B can be introduced either during or after the gelatin coating onto a solid support.
Coating methods are broadly described by Edward Cohen and Edgar B. Gutoff in Chapter 1 of xe2x80x9cModern Coating And Drying Technologyxe2x80x9d, (Interfacial Engineering Series; v.1), (1992), VCH Publishers Inc., New York, N.Y. In general, a fluid coating composition contains a binder, a solvent to dissolve or suspend the components, and optional additives such as surfactants, dispersants, plasticizers, biocides, cross-linking agents for toughness and insolubility, and conductive materials to minimize static buildup. All the components are mixed and dissolved or dispersed, and the coating fluid is sent to an applicator where it is applied to a substrate by one of several coating techniques. Heat is then applied to the coating to evaporate the solvent and produce the desired film, or the coating is solidified by the action of ultraviolet radiation or an electron beam.
The most suitable coating methodxe2x80x94including the coating speedxe2x80x94will depend on the quality and functionality desired and the materials being used, e.g., the substrate, the solvent, weight and viscosity of the coating, etc. For a single layer format, suitable coating methods may include dip coating, rod coating, knife coating, blade coating, air knife coating, gravure coating, forward and reverse roll coating, and slot and extrusion coating.
Coating speed can also be an important determinant in the choice of coating method. Although most methods can be used at low speeds, and all methods have a limiting upper speed, some work better at higher speeds. Curtain coating requires a minimum flow to maintain the integrity of the curtain. Therefore, this method is limited to higher speeds id a thin coating is to be obtained. In slide coating of multiple layers, interfacial instabilities are more likely to occur on the slide when the layers are very thin. Higher speeds, with their higher flows and thicker layers on the slide, tend to avoid these instabilities. See, p. 12, xe2x80x9cModern Coating and Drying Technologyxe2x80x9d, supra.
The gelatin has a laydown of 0.2 to 100 grams per square meter; preferably 10 to 50 grams per square meter.
Any well known coating method, such as bead coating or curtain coating, can be used to prepare the gelatin substrate. The gelatin could be coated with any other coating aids such as surfactants and thickeners to adjust its physical property. The gelatin used in the invention may be chemically modified either before, during or after the coating process to create more chemical functionalities that can react or interact with biologically active molecules or assemblies intended to be attached on this substrate.